Summary: ATP can be dispensable
for prespliceosome formation in yeast
Rhonda Perriman and Manuel Ares, Jr.1
Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz,
Santa Cruz, California 95064 USA
The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the
prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts
lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA
requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that
they are a functional intermediate in the spliceosome assembly pathway. ATP-independent
prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2
does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent
prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those
observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA
and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by
brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure.
[Key Words: Pre-mRNA splicing; spliceosome assembly; prespliceosome; U2 snRNA; CUS2; PRP5]
Received September 15, 1999; revised version accepted November 17, 1999.
The spliceosome is an elaborate ribonucleoprotein ma-
chine that recognizes and removes introns from premes-